Interpretive Summary: Thiamethoxam is a new neonicotinoid insecticide. Its thermolability and high polarity make it difficult to analyze by GC or GC-MS, requiring derivatization of the analytes prior to analysis. We have previously reported a polyclonal antibody-based immunoassay. In this study, we produced five monoclonal antibodies specific to thiamethoxam. In addition, we used these monoclonal antibodies to develop an automated kinetic exclusion fluorescent assay (KinExA) for the quantitative analysis of thiamethoxam. KinExA systems are used primarily to determine true liquid-phase equilibrium dissociation constants and association rate constants of antibodies by measuring the amount of antibodies in an equilibrium state with ligands in solution phase.
Limited use of KinExA was explored for quantitative analysis. The KinExA assay developed in this study has showed an I50 and the limit of detection of 30 ng/L and 16 ng/L of thiamethoxam, respectively.

Technical Abstract:
An ultra sensitive automated flow fluorescent immunoassay was developed using the KinExATM 3000 system for quantitative analysis of the neonicotinoid insecticide thiamethoxam. Five monoclonal antibodies were obtained and screened with a competitive ELISA. One monoclonal antibody designated as E6VI was evaluated for sensitivity, selectivity and solvent tolerance with the KinExA. Sensitivity determined from the concentration of half-maximal inhibition (I50) was obtained by plotting KinExA signals to a four parameter sigmoidal curve as a 14 function of analyte concentrations. For the most sensitive clone, the I50 and the limit of detection were approximately 30 ng/L and 16 ng/L, respectively. Cross-reactivity was estimated by measuring the equilibrium constants (Kd) for four other neonicotinoid insecticides (clothianidin, imidacloprid, dinotefuran, and acetamiprid). E6VI was very specific to thiamethoxam with <0.11% cross activity for tested neonicotinoids. An excellent correlation (r2=0.99) was obtained between spiked and measured concentrations of thiamethoxam in stream water.